Overview

According to data from the United Network for Organ Sharing (UNOS), more than 400,000 renal transplantations have been performed since the first successful renal transplantation in the 1950s. With improved surgical techniques and immunosuppressive medications, outcomes after renal and extrarenal (liver, pancreas, lung, heart) transplantation have continued to improve.

Most solid-organ transplantations are now performed as the therapeutic option of choice. In many cases, transplantation offers definitive treatment for a given disease entity. As a result, the list of indications for solid-organ transplantation has expanded considerably, placing increasing pressure on an already limited supply of donor organs.

The growth in the number of patients wanting or waiting for a transplant has outpaced the supply of available organs. As of April 2004, 96,060 patients were awaiting deceased donor organ transplantation in the United States, compared with half as many in 1995.
[1, 2] The UNOS reported that, as of July 2000, 45,600 people on its national patient list were waiting for a kidney.
[3] The average waiting time for a deceased donor kidney transplant is approaching 3 years, and that for heart transplants 18 months.
[2] Each year, more patients are placed on the waiting lists than receive transplants, causing the waiting time to increase. With such constraints, preservation of organs for transport between centers becomes crucial.

The technology of organ preservation has improved considerably. Several organ-preservation solutions are available, and these are being constantly modified to provide improved organ storage and outcomes.

This article discusses the pathophysiology, techniques, and principles of organ preservation, and it describes various preservation solutions currently used for kidney, liver, pancreas, small-bowel, lung, and heart transplantations.

Next:

Pathophysiology of Organ Preservation

The removal, storage, and transplantation of a solid organ from a donor profoundly alters the homeostasis of the interior milieu of the organ. These effects manifest in the degree to which the return of normal organ function is delayed or prevented after transplantation is completed. The injury an organ sustains during recovery, preservation, and transplantation occurs primarily as a result of ischemia and hypothermia. Techniques for organ preservation serve to minimize this damage to promote optimal graft survival and function.

Phases of Organ Damage During Transplantation

Damage to organs during transplantation occurs in 2 phases.

The first, the warm ischemic phase, includes the time from the interruption of circulation to the donor organ to the time the organ is flushed with hypothermic preservation solution. In multiorgan recovery, the organs are cooled before they are removed.

The second, the cold ischemic phase, occurs when the organ is preserved in a hypothermic state prior to transplantation into the recipient.

Mechanisms of Tissue Injury

Integrity of the cell structure

The cell membrane plays a structural role for the cell and provides an active interface with the extracellular environment. Receptors, ion regulation, and enzyme systems linked to the cell-membrane complex contain extracellular, transmembrane, and intracellular components essential to their function.

The stability of the membrane to chemical and water permeability depends on the integrity of the lipid bilayer and on tight control of temperature, pH, and osmolarity.
[4] Organ ischemia and preservation disrupt all these relations. Lowering the temperature causes a phase transition of lipids and results in profound changes in membrane stability. In addition, it drastically alters the function of membrane-bound enzymes.
[4] Hypothermia-induced structural changes in the membrane increase permeability, which contributes to cell swelling. Therefore, organ-preservation solutions are hypertonic to minimize these alterations.

Ionic composition of the cell

The sodium-potassium adenosine triphosphatase (Na-K ATPase) pump functions to maintain the ionic composition of the cell. The pump is disrupted because of the lack of adenosine triphosphate (ATP) production and by excessive production of hydrogen ions because of anaerobic metabolism during ischemia. When the sodium-potassium ATPase pump is paralyzed, potassium moves out of the cell and diffuses down its concentration gradient to the extracellular space, whereas sodium, which is normally kept at a low concentration in the cell, pours in. This ionic shift causes cell swelling and disruption of the cell if unchecked. Current preservation solutions have electrolyte compositions similar to the milieu inside the cell, with high potassium and low sodium concentrations to minimize the osmotic gradients.

Hydrogen-ion production continues in ischemic organs and causes intracellular pH to decrease without replenishment of buffering capabilities. Under conditions requiring a switch from aerobic to anaerobic glycolysis, the production of lactic acid also increases. The liver appears to be especially susceptible to this type of injury.

Endothelin, a 21–amino acid peptide with potent vasoconstrictor properties, is another factor that plays a major role in ischemia by inducing vasospasm, which delays recovery of organ function after revascularization.
[5, 6] Investigators have evaluated the use of endothelin A and B receptor antagonists to improve results after solid-organ transplantation, reporting favorable results.

ATP generation

A combination of the anaerobic enzymatic breakdown of glucose (glycolysis) and aerobic cellular respiration provides for the energy requirements of aerobic cells. These processes encompass the transfer of electrons from organic molecules to molecular oxygen. Hypothermia decreases the metabolic rate and slows enzymatic degradation of cellular components, but metabolism is not completely suppressed. Cooling from 37°C to 0°C reduces cellular metabolism 12-fold.
[4] Although metabolism and utilization of cellular energy stores are slowed, ATP and adenosine diphosphate (ADP), the major sources of cellular metabolic energy, are gradually depleted during hypothermia. This depletion is due to residual energy requirements of the cell that exceed the capacity of the cell to produce ATP.

During ischemia and organ preservation, the glycolytic pathway is shunted to lactate production with generation of 2 ATP, as the Krebs tricarboxylic acid cycle (TCA) cycle and mitochondrial respiration are impaired. Therefore, mitochondrial dysfunction is responsible for most of the changes in cellular energy associated with ischemia and organ preservation.

Hypothermic preservation reduces the activity of mitochondrial enzymes.
[7] Cellular respiration, which requires adenine nucleotide substrates, is reduced. For ADP to be transported into the mitochondrion as a substrate for conversion to high-energy ATP, a membrane adenine nucleotide translocase is required. Hypothermia impairs the function of the translocase because amounts of ADP available in the mitochondria for conversion to ATP are decreased.
[8] Phospholipid hydrolysis by phospholipases increases levels of free fatty acids, which also affect function of the translocase. Adenylate kinase converts excess ADP accumulating in the cytoplasm to ATP, and adenosine monophosphate accumulates as a by-product. This effect reduces purine synthesis and results in the loss of ATP precursors from the cell.

Vascular renal resistance during hypothermic machine perfusion has been found to be an independent risk factor for 1-year graft failure. Renal resistance must be taken into account when evaluating graft quality and predicting a successful outcome; however, renal resistance on its own cannot be used to precisely predict the outcome.
[9]

Reperfusion injury

Much of the injury to transplanted organs occurs not during ischemia, but during reperfusion. This finding has led to many advances in organ preservation aimed at preventing this type of injury. Furthermore, some of the events that occur during reperfusion may result in enhanced immunogenicity of the graft.
[10] Oxygen free-radicals generated during reperfusion are the main cause of the reperfusion injury, but cytokines and nitric oxide also play a role.
[10, 11]

Oxygen free-radicals are the most important mediators of reperfusion injury and act by means of various mechanisms. During ischemic states, increased intracellular calcium levels activate cytosolic enzymes, resulting in the conversion of xanthine dehydrogenase to xanthine oxidase.
[10] Both xanthine dehydrogenase and xanthine oxidase catabolize hypoxanthine and xanthine to uric acid, and xanthine oxidase uses molecular oxygen as an electron acceptor, forming superoxide as a result.
[10] Superoxide anion rapidly reacts with itself to form hydrogen peroxide, a potent oxidant capable of injuring the cell by oxidizing lipid membranes and cellular proteins. Hydrogen peroxide then produces a cascade of oxygen free-radicals, including hydroxyl radical and singlet oxygen, that are even more potent than the others. The damaging effects of oxygen free-radicals begin on reperfusion of the organ.

During ischemic conditions, tissue oxygen levels fall below the threshold needed to allow xanthine oxidase to metabolize xanthine and hypoxanthine. This decrease allows the intracellular concentration of these metabolites to rise. On reperfusion, oxygen is suddenly available, and metabolism proceeds rapidly, resulting in a sudden production of reactive oxygen intermediates. The cellular pathways to scavenge oxygen free-radicals are overwhelmed, and cellular injury ensues.

Allopurinol, an inhibitor of xanthine oxidase, has protective effects when it is used before the ischemic insult, as shown in various experimental systems. Lipid peroxidation is another consequence of free radical generation.
[11] In this process, interaction of highly reactive oxygen species with polyunsaturated fatty acids in the cell membrane starts a chain reaction that may ultimately destroy cellular integrity and result in cell death. The magnitude of lipid peroxidation appears to be inversely related to levels of glutathione, which functions as an endogenous free-radical scavenger.
[11] Therefore, glutathione and other agents that protect against peroxidation are useful in organ preservation solutions to attenuate reperfusion injury.

Production of oxygen free radicals also initiates production of prostaglandins, including leukotriene B4, by means of direct activation of phospholipase A210. This chemoattractant causes leukocyte adherence to vascular endothelium. These neutrophils may contribute to local injury by plugging the microcirculation and by degranulation with resulting proteolytic damage to the organ.

Cytokines are intercellular messenger molecules that may be produced in various normal and pathophysiologic states. Ischemia and reperfusion are associated with marked release of tumor necrosis factor (TNF)-alpha, interferon-gamma, interleukin-1, and interleukin-8.
[12] These cytokines cause upregulation of adhesion molecules and cause leukocyte adherence and platelet plugging after revascularization, resulting in graft failure and rejection.

Nitric oxide (NO) is an extremely labile autocoid generated by nitric oxide synthase from L-arginine. Reports indicate that NO production is induced by inflammatory cytokines, such as TNF-alpha, interferon-gamma, and interleukin-1. Increased NO synthesis also correlates with acute rejection.
[13]

Previous

Next:

Preservation Solutions and Their Pharmacology

Various flush solutions are used for organ preservation. Each substantially differs in their composition, but the purposes of each are similar: to prevent cellular edema, to delay cell destruction, and to maximize organ function after perfusion is reestablished.
[14, 15]

Euro-Collins solutions

The development of solutions of intracellular electrolyte composition allowed for organ preservation to be attempted. These early solutions, called Collins solutions, contained high concentrations of potassium, magnesium, phosphate, sulphate, and glucose. Euro-Collins solution was developed as a modification of the original Collins solution and contained high concentrations of potassium (110 mM), phosphate (60 mM), and glucose (180 mM). Organ preservation improved with the use of Euro-Collins solution. When it was used for kidney preservation, delayed renal function after implantation was significantly reduced. The solution was adequate for use in preserving the heart, liver, and lung.

Ross-Marshall citrate solutions

Ross-Marshall citrate solutions were developed as alternatives to the Collins solutions. Their electrolytic compositions are similar except that citrate replaces phosphate, and mannitol replaces glucose. The citrate acts as a buffer and chelates with magnesium to form an impermeable molecule that helps stabilize the extracellular environment. It is not commonly used in clinical practice.

Bretschneider histidine tryptophan ketoglutarate solution

Initially developed as a cardioplegia solution for the use in open heart surgery, Bretschneider histidine tryptophan ketoglutarate (HTK) solution was found to be effective in liver and kidney preservation. Its contents include histidine (200 mM), mannitol (30 mM), tryptophan and alpha-ketoglutaric acid. It also contains low concentrations of sodium, potassium, and magnesium. Histidine serves as a buffer, and tryptophan, histidine, and mannitol act as oxygen free-radical scavengers. The solution improved renal function after transplantation compared with Euro-Collins solution. It has become increasingly popular in recent years, but some concern exists regarding an increased risk for pancreatitis when the pancreas graft is preserved with HTK solution. This has not been confirmed in randomized studies.

Phosphate-buffered sucrose solution

This solution contains sucrose 140 mmol/L and sodium hydrogen and dihydrogen phosphate as buffers. In experimental studies, it preserved dog kidneys for 3 days. It is not commonly used today.

University of Wisconsin solution

University of Wisconsin (UW) solution was developed for liver, kidney, and pancreas preservation. It has been considered the standard for renal and hepatic preservation, effectively extending the ischemic time for kidneys and livers and allowing them to be transported considerable distances to waiting recipients. UW solution has also been successfully applied to small-bowel and heart preservation.

The composition of the solution is complex. Analysis of its various components has shown that some may be omitted or replaced with results similar to that of the original solution.

The solution has an osmolality of 320 mmol/kg and pH 7.4 at room temperature and is composed of the following:

Potassium 135 mmol/L

Sodium 35 mmol/L

Magnesium 5 mmol/L

Lactobionate 100 mmol/L

Phosphate 25 mmol/L

Sulphate 5 mmol/L

Raffinose 30 mmol/L

Adenosine 5 mmol/L

Allopurinol 1 mmol/L

Glutathione 3 mmol/L

Insulin 100 U/L

Dexamethasone 8 mg/L

Hydroxyethyl starch (HES) 50 g/L

Bactrim 0.5 ml/L

HES conveys no advantage to the solution when used for cold storage, and it, in fact, adds to the viscosity of the solution as well as to the expense. HES-free derivatives of the solution have given similar, if not better, clinical results than those of the original formulation.

The lactobionate is the major effective component of the solution. Its insoluble nature maintains the colloid oncotic pressure of the solution, delaying or preventing equilibration of the solution across the cell membrane, and thus delaying the development of cellular edema.

The lowered concentration of potassium improves the flushing efficiency of the solution by removing the vasoconstrictive effect of the high potassium solution.

Glutathione is unstable in solution and effective as an oxygen free-radical scavenger only if it is added immediately before use. Adenosine and allopurinol help in this function.

Celsior solution

Celsior is a recently developed extracellular-type, low-viscosity (due to the absence of HES) preservation solution that couples the impermeant, inert osmotic carrier from UW solution (by using lactobionate and mannitol) and the strong buffer from Bretschneider HTK solution (by using histidine). The reduced glutathione in Celsior solution is the best antioxidant available. The solution was specifically designed for heart transplantation. It is being currently used in clinical lung, liver, and kidney transplantations and it is under investigation for pancreas transplantation.

The contents of Celsior solution are as follows:

Sodium 100 mmol/L

Potassium 15 mmol/L

Magnesium 13 mmol/L

Calcium 0.25 mmol/L

Lactobionate 80 mmol/L

Glutathione 3 mmol/L

Glutamate 20 mmol/L

Mannitol 60 mmol/L

Histidine 30 mmol/L

Kyoto ET solution

Researchers at Kyoto University developed a new solution that contains a high sodium concentration, a low potassium concentration, trehalose, and gluconate. The solution is chemically stable at room temperature. It is being investigated for skin storage and lung preservation in a rat model. ET Kyoto solution is also being actively investigated in clinical trials for transplantation of the lungs, heart, and other organs.

Its constituents include the following:

Sodium 100 mmol/L

Potassium 44 mmol/L

Phosphate 25 mmol/L

Trehalose 41 mmol/L

HES 30 gm/L

Gluconate 100 mmol/L

Previous

Next:

Techniques of Organ Preservation

Because most transplanted organs are from deceased donors, the organ must inevitably be stored after its removal from the donor until it can be transplanted into a suitable recipient. The donor and recipient are often in different locations, and time is needed to transport the donor organ to the hospital where the recipient is being prepared for transplantation. Effective, safe, and reliable methods are needed to preserve the organ ex vivo until transplantation can be performed. Acceptable preservation times vary with the organ. Most surgeons prefer to transplant the heart within 5 hours of its removal; the kidney can safely be stored for 40-50 hours, but earlier transplantation is preferred. Most pancreas transplants are performed after 5-15 hours of preservation. Liver transplantations usually are performed within 6-12 hours.

Hypothermic preservation

Hypothermia is the preferred technique of organ preservation because it is simple, does not require sophisticated expensive equipment, and allows ease of transport. Hypothermia is beneficial because it slows metabolism.
[4, 16, 17] Organs exposed to normothermic ischemia remain viable for relatively short periods, usually less than 1 hour.

In warm ischemia, the absence of oxygen leads to a rapid decline in ATP levels in the cells, to a redistribution of electrolytes across the cell membrane, and to a decrease in biosynthetic reactions. However, biodegradable reactions continue; these include the accumulation of lactic acid, a decrease in intracellular pH, proteolysis, lipolysis, and lipid peroxidation.

With hypothermia, the degradative reactions are considerably slowed but not halted. A 10 º C decrease in temperature slows the metabolic rate approximately by a factor of 2. Cooling an organ from 37 º C to approximately 0 º C slows metabolism by a factor of 12-13. Hypothermia alone is not sufficient for adequate preservation because of the time necessary for optimal use of deceased donor organs. Therefore, the organ must also be flushed with an appropriate preservation solution.

Two techniques of hypothermic preservation are used: simple cold storage and continuous hypothermic perfusion. With simple cold storage, the organ is flushed with cold preservative solution and placed in a sterile bag immersed in the solution. The sterile bag is placed inside another bag that contains crushed ice. Advantages of simple cold storage include universal availability and ease of transport. With continuous hypothermic perfusion, which Belzer developed in 1967, a machine is used to continuously pump perfusion fluid through the organ.
[18] In this way, oxygen and substrates are continuously delivered to the organ, which maintains ion-pump activity and metabolism, including the synthesis of ATP and other molecules.

For kidneys, machine perfusion offers superior results compared with simple cold storage.
[19] With simple cold storage, approximately 25-30% of transplanted kidneys have delayed graft function, but with machine perfusion, the rate can be less than 10%. The perfusate is similar to the UW solution, except for the impermeant. For continuous perfusion, gluconate is used in place of lactobionic acid.
[18]

Freezing and thawing

Cryopreservation has long fascinated scientists, who have made many attempts at cryopreserving organs and even full bodies by means of conventional freezing and thawing.
[20] These efforts usually focused on simple transferring techniques that have worked for cell preservation to organs.

Notable problems are associated with this approach. For instance, the packing density of cells in an organ can approach 80%, but preservation of isolated cells becomes technically difficult at a cell concentration above 20%.
[21] In addition, the presence of many different cell types, each with its own requirements for optimal cryopreservation limits the recovery of each when a single thermal protocol is imposed on all cells. Moreover, extracellular ice can cause mechanical damage to the structural integrity of the organ, particularly the vascular component, where ice is likely to form.
[20] Mechanical fractures occur in the vitreous solids that exist between ice crystals when thermal stresses occur at low temperatures. These fractures separate parts of the organ from each other. The attachments that form between cells and between cells and their basement membranes are disrupted. Last, osmotic movement of interstitial water causes mechanical stresses.

Each of the problems is a formidable source of damage, above those already well known from studies of cells in suspension. Cryopreservation of whole organs is not possible using current technology.

Vitrification

Perhaps the most promising approach to the cryopreservation of whole organs lies with the process of vitrification.
[21] This is the process of taking an aqueous solution and making it into an amorphous solid.

A liquid can be cooled past its melting point without a phase change. The formation of ice first requires nucleation, a stochastic process by which an ice nucleus (cluster of water molecules that reaches a critical size) forms spontaneously. With lowered temperatures, the critical size of a nucleus decreases and eventually approaches the size of the clusters in the liquid. The solution can be cooled to the homogeneous nucleation temperature (temperature at which the probability of nucleation equals 1) in a supercooled state, but below this point crystallization occurs. The supercooled liquid exists in a metastable state until the transition temperature is reached. At this temperature, the time required for the molecular translations needed in nucleation or crystalline growth becomes infinite; therefore, the amorphous solid is stable below this point.

Vitrification can also be achieved by adding solutes that develop a structure in water that must be broken down for crystalline growth. The solute molecules impede this growth simply by blocking other water molecules and interfering with hydrogen bonding necessary for ice formation. Both the kinetic approach and the solute approach are additive. Therefore, as the solute concentration is increased, the cooling rate necessary to achieve vitrification is lowered.

The problem with vitrifying organs is that about half of the water in an organ must be replaced with solute molecules for vitrification to occur at reasonable cooling rates. The difficulties in developing successful vitrification techniques include infusing high concentrations of vitrification solutes into the organ and removing them on thawing and preventing fracturing of the organs during cryogenic storage and warming the organs fast enough to prevent devitrification, among other problems. Vitrification is not currently being used in clinical transplantation.

Principles of Organ Preservation

Preservation of the organ begins when a donor is identified, and the donor's hemodynamic status must be adequately maintained to prevent organ injury before its recovery and preservation. An organ can be injured because of cardiovascular instability and hypotension. During the operation to remove the organ, the warm ischemia time must be minimized, and the organ must be cooled rapidly in situ or by means of well-timed back-table flush out.
[22] Thereafter, the organ is maintained in hypothermic state and transported in this state until it is ready to be transplanted. Cold ischemia time must be kept within the limits prescribed for the particular organ.

Donor Maintenance

Tissue perfusion

Because most donors are treated initially with a minimum of fluids to prevent increased cerebral edema, one of the first priorities in donor maintenance is the prompt restoration of intravascular volume. Volume resuscitation with 3-10 L may be required, and pressor agents may be needed to support blood pressure.
[22]

Crystalloids may be used, and lactated Ringer's solution is often given because its low sodium concentration counteracts the sodium-increasing effects of diabetes insipidus. If the latter condition has resulted in severe hypernatremia, free water may need to be added, and desmopressin may also be used selectively in severe cases. Colloid and blood should be used to restore and maintain osmotic pressure and normovolemia.

The hematocrit should be kept at approximately 35% by volume to replace traumatic losses and maintain oxygen-carrying capacity. Enough volume should be administered to achieve a urine output of at least 100 mL/h and a systolic arterial pressure of 90-120 mm Hg. Arterial blood pressures higher than this are usually unnecessary because of the loss of sympathetic tone that accompanies brain death.

Oxygenation and ventilation

Arterial blood gases should be checked regularly, and the ventilator should be adjusted to optimize gas exchange and acid-base balance. The oxygen supply should be adequate to maintain an arterial oxygen saturation of greater than 95%, and, if available, a mixed venous oxygen saturation above 70%. Low levels of positive end-expiratory pressure may facilitate balance of the oxygen supply and demand.

Inotropic support

Most donors are hypovolemic at brain death and are receiving inotropic support in lieu of volume to maintain their blood pressure. Dopamine hydrochloride is the most commonly used agent. High doses of dopamine maintained for long periods before organ recovery may be associated with increased rates of acute tubular necrosis and hepatic allograft failure. Cardiac recovery is often abandoned if high doses of inotropic support are required despite adequate volume status.

Other inotropic agents, such as isoproterenol, epinephrine, norepinephrine, and phenylephrine, are occasionally used, but use of these agents should be discouraged because of their peripheral vasoconstrictive effects. Dobutamine is occasionally used in conjunction with low doses of dopamine.

Prevention of hypothermia

Thermoregulatory homeostatic mechanisms are destroyed with brain death, and severe hypothermia may lead to ventricular arrhythmia and cardiac arrest. Warming blankets should be placed above and below the donor to keep the body temperature above 35 º C. If maintenance of normal body temperature is problematic, intravenous fluids may be prewarmed before their administration, and a heated humidifier circuit can be added to the ventilator.

Hypothermia and the composition of the organ-preservation solution are key factors in successful organ preservation. In the cold storage of organs, the organ is rapidly cooled to approximately 4 º C by flushing out the vascular system with an appropriate organ-preservation solution; this is the Starzl method of rapid cooling. The flushing should remove the blood as completely as possible, and the solution be delivered at a pressure that is not damaging to the organ (usually 60-100 cm H2 O) and in a volume that is not excessive. The volume used for each organ varies, but the liver usually is flushed with approximately 2-3 L, the kidney is flushed with 200-500 mL, and the pancreas with 200-500 mL. The organ is then placed in a sterile container and kept cold at 4-6 º C.

Multiple-Organ Recovery

After brain death is declared, the deceased is identified as a suitable organ donor, and the next of kin gives permission, the organs must be recovered. Techniques of multiple organ recovery allow for the removal of the heart, lungs, kidneys, liver, intestine, and pancreas from a single donor for transplantation into 6 or more recipients.

The heart-beating donor is brought to the operating room from the intensive care unit or emergency department, with appropriate hemodynamic and electrocardiographic monitoring. Oxygen is delivered by means of manual bag-valve mask with 100% inspired concentration. Inotropic drug infusions are continued during transport and recovery. The anesthesiologist should maintain close communication with the recovery teams to prevent cardiac arrhythmia, hypotension, and hypoxemia.

Steps in the procedure can be categorized as follows: (1) incision, (2) exploration and inspection, (3) mobilization of individual organs, (4) in situ perfusion, (5) removal of organs, and (6) closure of the incision. Postrecovery processing, packaging, and transport to the recipient centers are the final steps.

Incision, exploration, and inspection

The entire torso is prepared with an iodine-containing solution, and a field is draped from the neck to the pubis. General exploration is carried out to confirm that unexpected conditions that preclude donation, such as tumor, infection, or specific organ damage, are absent. A complete midline incision from suprasternal notch to pubis is made for multiple-organ recovery. The sternum is split. If necessary, cruciate abdominal incisions are added to facilitate exposure of the intra-abdominal organs.

Mobilization of individual organs and in situ perfusion

In the rapid technique of organ procurement, little mobilization of individual organs is necessary. Preparation for in situ perfusion consists of a complete Kocher maneuver with a Cantrell maneuver. The aorta is exposed from the bifurcation distally to the level of the left renal vein proximally. The inferior mesenteric artery is divided. Lumbar branches may be ligated and divided at this point. The inferior vena cava is similarly exposed.

Next, the left triangular ligament of the liver is taken down, exposing the crural muscle at the aortic hiatus. The aorta is encircled with tape at the diaphragm. In situ perfusion through the distal aorta and cross-clamping of the proximal aorta begin immediately in the event the donor becomes hemodynamically unstable or sustains cardiac arrest. In situ perfusion prevents warm ischemia. Stability of the donor allows for further preparation before in situ flushing.

The inferior mesenteric vein is isolated as it enters the retroperitoneum behind the pancreas. This vein provides convenient access for in situ portal flushing by means of a small catheter, such as a Javid shunt. The pars flaccida of the lesser omentum should be inspected for evidence of a replaced left hepatic artery arising from the left gastric artery, and minimal dissection of the hepatic artery is necessary.

The course of the ureters should be identified. Gerota's fascia is widely incised to allow topical cooling with iced slush solution to supplement the in situ perfusion. Complete mobilization of the kidneys before in situ flushing is unnecessary and poses a risk of damage to the renal vessels or inadvertent division of accessory renal arteries.

Removal of organs

A team working simultaneously with the abdominal-retrieval team can prepare the heart and lungs for removal. The superior and inferior vena cava are mobilized, and the aortic arch is dissected sufficiently for the placement of a crossclamp at the time of infusion of aortic root cardioplegia. Preliminary mobilization of the lungs is usually performed, and access to the pulmonary artery is necessary for pulmonary preservation. When all teams are ready, a coordinated sequence of events ensures that all organs are simultaneously cooled and protected.

The donor is given systemic heparin. A Javid shunt is advanced through the inferior mesenteric vein near the pancreas and advanced into the portal vein. A cardioplegia needle is positioned in the aortic arch. The distal abdominal aorta is cannulated for in situ perfusion of the kidneys, liver, and pancreas, and an exsanguination cannula is placed in the distal inferior vena cava. As an alternative, the inferior vena caval-right atrial junction may be divided in the chest with suction catheters placed in the caval lumen and right thoracic cavity to decompress the venous circulation at the moment of aortic cross-clamping.

Another cannula is placed in the distal vena cava for venous decompression and exsanguination. The aortic arch and the abdominal aorta at the diaphragm are simultaneously cross-clamped. (In the case of simultaneous liver/small bowel allograft procurement, the supraceliac aorta is not clamped because of the need to preserve the donor thoracic aorta with the graft without intimal injury from the clamp).

Cardioplegia solution is infused under pressure into the aortic root, perfusing the coronary arteries and arresting the heart. Ventilation is ceased. Portal and distal aortic perfusion are initiated with ice-cold preservation solution. Topical iced slush is placed in the abdomen and chest to assist the cooling process. The heart and lungs are then removed. The clamp is removed from the distal vena caval cannula for exsanguination and to preclude venous congestion.

The liver and pancreas and/or intestine are removed en bloc. The diaphragm surrounding the suprahepatic inferior vena cava and adjacent to the bare area of the liver is divided. The infrahepatic inferior vena cava is divided just cephalad to the left renal vein. The liver, pancreas and intestine unit is now attached by only the distal superior mesenteric vessels coursing to the small bowel and by the aortic origins of the superior mesenteric artery and celiac axis. The liver, pancreas, and intestine are separated as a bench procedure; the celiac axis is retained with the liver.

The process of separation depends, at this point, on the allocation of organs. If the intestine and pancreas have been placed separately, the superior mesenteric arterial and venous branches emerging from the uncinate process are divided, ligating the pancreatic side. If the liver/small bowel graft has been allocated to the same recipient, no further dissection is performed. If only the liver and pancreas have been allocated, the superior mesenteric artery and vein are divided distal to the pancreas (often with a vascular load from a stapling device), and the cylinder of aorta between the superior mesenteric artery and celiac axis is divided.

The splenic artery is divided at its origin. This vessel and the superior mesenteric artery are reconstructed with a bifurcated donor iliac artery graft. The gastroduodenal artery is ligated and divided. The portal vein is divided approximately 1 cm from the superior edge of the pancreas, and the common bile duct is divided just superior to its entrance into the pancreas.

The kidneys are also removed en bloc. The ureters are mobilized with a generous amount of periureteral tissue to avoid devascularization and are divided near their entrance into the bladder. Dissection is carried out posterior to the aorta and vena cava in the plane of the prevertebral fascia. Once removed, the left renal vein is divided with a small cuff of vena cava. The aorta is opened in the midline to identify the orifices of the renal arteries from within the lumen. In this way, multiple renal arteries can be readily identified and kept on a single aortic (Carrel) patch.

Closure of the incision

After the abdominal and thoracic organs are removed, a sampling of lymph nodes and spleen is taken for tissue typing and crossmatch testing. The chest and abdomen are closed, and standard postmortem care is given. If the intestine has been allocated, use of antilymphocyte globulin to avoid graft versus host disease from the lymphoid-rich bowel graft is perfused in the donor. Lymph nodes should be procured prior to initiation of the antilymphocyte globulin because difficulty in collecting adequate viable lymphocytes for the crossmatch has been noted in the past when they are procured after procurement of the solid organs.

Preservation Solutions

Two requirements of any ideal preservation solution are (1) impermeant molecules that suppress hypothermically induced cell swelling and (2) an appropriate biochemical environment. Impermeants are agents that remain outside the cells and that are sufficiently active osmotically to retard the accumulation of water by the cell. Constituents of an ideal solution and their individual function are detailed below and in Preservation Solutions and their Pharmacology above.

Previous

Next:

Current Preservation Techniques

Kidney, liver, and pancreas

Until relatively recently, the primary solution used for cold-storage preservation of the kidneys was Euro-Collins solution.
[23] Its formulation provides a hyperosmolar environment with an intracellular electrolyte composition intended to reduce cellular swelling. In combination with hypothermia, kidneys can be safely stored in this solution for up to 36-48 hours before transplantation.
[23]

In the 1980s, the advent of new immunosuppressive agents, such as cyclosporine, meant that, for the first time, extrarenal organs could be transplanted with good success rates. With this development, the need for effective preservation became apparent.

The limitations of cold ischemia to approximately 8 hours for the liver and pancreas meant that donor and recipient teams had to be closely coordinated.
[24] Complex recipient operations that required a multitude of ancillary support services had to be organized in the middle of the night, and all personnel involved in the procedure, including the surgeons, were starting the operation in a suboptimal fatigued condition.

Researchers at the University of Wisconsin developed a solution that simplified the practice of hepatic and pancreatic transplantation at most centers in North America and Europe.
[3] The solution, UW cold-storage solution, is based on lactobionate, raffinose, HES, and a host of other ingredients designed to provide high-energy phosphate precursors, hydrogen-ion buffering capacity, and antioxidant properties.
[24]

Lactobionate, an impermeant anion to prevent cellular swelling, was used in place of the glucose contained in Euro-Collins solution. Raffinose, a naturally occurring trisaccharide of fructose, glucose, and galactose, provides additional osmotic activity; and HES is a colloid intended to prevent an increase in the extracellular space, though it makes the solution viscous.

Most teams that perform hepatic and pancreatic transplantation prefer UW solution as the preservation method of choice.
[24] Both the liver and pancreas can reliably be stored for 12-18 hours, and isolated clinical cases with total cold ischemia times in excess of 30 hours have been reported.
[25] However, evidence is accumulating that cold ischemia times longer than 12 hours may be associated with a high incidence of biliary strictures.
[25]

Whether the UW cold-storage solution has any significant advantages over Euro-Collins solution for the preservation of kidneys is unclear. In a large, randomized, multicenter European trial, patient and graft survival rates were similar among recipients of the 2 solutions, but the incidence of delayed graft function requiring dialysis was reduced by approximately one third in the UW group. Results of ongoing trials are needed before a definitive statement can be made about the relative merits of the 2 solutions in renal preservation.

New preservative solutions, including Celsior solution, are emerging and may have advantages over UW solution,
[25, 26] though results are conflicting.
[27] The Kyoto solution was evaluated for kidney storage and found to be as good as UW solution. Furthermore, the Kyoto solution was less viscous and stable at room temperature for as long as 3 years, and it was cost effective as compared with UW solution.
[28]

Another solution that is being increasingly used presently is Bretschneider's HTK solution (Custodiol). Originally developed for cardioplegia, HTK is routinely used for liver, kidney, and heart transplantation. HTK solution has been shown to be superior to Euro-Collins solution in prospective trials (Ringe, 2006; Agarwal, 2006; Nardo, 2005; Mangus, 2006). Advantages include lower viscosity and less leucocyte adhesion. In the European multicenter trial, HTK was shown to be equivalent to UW solution except for superior delayed graft function rates in the HTK group. Other reports have shown better delayed graft function rates with UW solution (Agarwal, 2005).

A randomized study comparing the efficacy of UW solution versus HTK on 102 liver transplant recipients reported that both solutions were equally effective in graft preservation.
[29] Mangus et al reported from their single center study of extended criteria liver donors that HTK and UW were clinically indistinguishable but that biliary complications were less common with HTK.
[30] However, in a retrospective single center study of living donor liver transplantation from Korea, the incidence of preservation injury was greater in grafts treated with HTK versus UW solution.
[31]

Islet transplantation has become a feasible treatment option for patients with type 1 diabetes mellitus after improvements were made in the diabetogenicity of immunosuppression and in the preparation of sufficient quantities of highly viable islets for transplantation. However, a minimum of 10,000 islet equivalents per kilogram of the recipient's body weight islet mass is required to achieve insulin independence,
[32] and long-term outcomes have been disappointing, with a 5-year insulin-independence rate of 8%.

UW solution has proven to be effective in pancreas preservation, leading to clinically successful whole-pancreas transplantation. In addition, human pancreatic grafts can be preserved for longer than 24 hours by using UW solution. However, in clinical islet transplantation, prolonged cold storage in UW solution before islet isolation significantly reduced recovery of viable islets.
[33]

A 2-layer cold-storage method using perfluorochemical (PFC) and UW solution has been evaluated for whole-pancreas preservation. This method continuously supplies sufficient oxygen to a pancreas during preservation and reduces cold ischemic injury by producing ATP, which maintains cellular integrity and controls ischemic cell swelling.
[33] Canine pancreata subjected to 90 minutes of warm ischemia were resuscitated during preservation by using the 2-layer method at 4°C for 24-48 hours.
[34] A prospective randomized study comparing HTK versus UW in 68 pancreas transplant recipients reported that the solutions were equally suitable for perfusion and organ preservation. In this study, 6-month patient survival was 96.3% (HTK) versus 100% (UW) (P = 0.397).
[35]

Heart and lungs

Cardiac preservation has changed relatively little in recent years. Hyperkalemic, crystalloid cardioplegia solution is used at 4°C, and 4 hours is the generally accepted limit of cold ischemia.
[36] For this reason, donor and recipient operations must be finely coordinated. Every effort is made to limit the ischemic time (the time between the initial interruption of coronary flow by means of aortic cross-clamping in the donor and the removal of the clamp in the recipient) to less than 4 hours. Although laboratory data and isolated clinical reports suggest that good results may be expected with storage times of 8 hours or longer with the use of the newer preservation solutions,
[36] increased ischemic time remains a strong and important independent risk factor for poor recipient outcome.

The maximal safe interval for the lung to remain ischemic, even when cooled, has not been defined. Based on empiric observation, 6 hours is the selected limit.
[37] This constraint limits the distance that can be traveled to recover donor lungs. The limits of donor-lung ischemia have been expanded because of efforts to develop bilateral, sequential lung replacement. The second lung to be implanted is ischemic for longer than the first because the lungs are not implanted simultaneously. The longest cold ischemic time was 9-10 hours, and the lung functioned well within 24 hours after implantation. Although donor-lung dysfunction occurs in 5-10% of cases, it is usually reversible. This problem is not correlated with prolonged donor-lung ischemic time.

Whether one type of preservation solution for lung allografts is better than another remains to be determined. The Celsior solution may be better for early lung function than the standard Euro-Collins solution.
[38] One clinical study demonstrated less reperfusion injury, better immediate and intermediate function, and better early and long-term survival for donor lungs preserved with Celsior than with Euro-Collins solution.
[37] Core cooling of the donor with an extracorporeal circuit has been used extensively in the United Kingdom for cardiopulmonary transplantation. Others have used an immersion technique without flushing the pulmonary artery. Hypothermia is applied, along with intracellular-type solutions. The Kyoto solution also appears promising in lung transplantation.
[39] The optimal preservation solution for the lungs remains unknown, and deficiencies in the quality and duration of preservation still limit the results of pulmonary transplantation.

Small bowel

The transplantation of segments of small bowel with or without concomitant liver transplantation is being performed with increased regularity as the treatment of choice for short-gut syndrome. The bowel is usually preserved with UW solution, though experimental evidence does not indicate that this solution offers any advantage over simple hypothermia.
[40] Experimental studies have also reported that ex vivo application of carbon monoxide in UW solution may also prevent reperfusion injury.
[41] The use of anti-oxidative agents has also shown promise in laboratory studies as an intervention to minimize preservation injury.
[42] The same investigators reported that this may occur due to down-regulation of pre-apoptotic and up-regulation of cytoprotective signals in the presence of a nutrient-rich preservation solution.
[43] Laboratory studies on Wistar rates have also reported better small bowel integrity and function compared to UW solution.
[44]

Most procurements of small bowel are from donors who have many useful organs. Therefore, standard intra-aortic preservation techniques are used. The current limit of cold ischemia time for small bowel is approximately 12 hours.
[45] Reports suggest that the use of luminal flush solutions, both UW and amino acid – enriched solutions, improve mucosal barrier function, as measured by means of mannitol permeability, and decrease villous morphologic injury.
[46]

Previous

References

UNOS. United Network for Organ Sharing Web page. United Network for Organ Sharing. Available at http://www.unos.org. Accessed: May 17, 2007.

Ad Hoc Committee of the Harvard Medical School. A definition of irreversible coma. Report of the Ad Hoc Committee of the Harvard Medical School to Examine the Definition of Brain Death. JAMA. 1968 Aug 5. 205(6):337-40. [Medline].